Although abnormal TGFβ signaling is observed in several heritable forms of thoracic aortic aneurysms and dissections including Marfan syndrome, its precise role in aortic disease progression is still disputed. Using a mouse genetic approach and quantitative isobaric labeling proteomics, we sought to elucidate the role of TGFβ signaling in three Fbn1 mutant mouse models representing a range of aortic disease from microdissection (without aneurysm) to aneurysm (without rupture) to aneurysm and rupture. Results indicated that reduced TGFβ signaling and increased mast cell proteases were associated with microdissection. In contrast, increased abundance of extracellular matrix proteins, which could be reporters for positive TGFβ signaling, were associated with aneurysm. Marked reductions in collagens and fibrillins, and increased TGFβ signaling, were associated with aortic rupture. Our data indicate that TGFβ signaling performs context-dependent roles in the pathogenesis of thoracic aortic disease.

Objective:Fragments of fibrillin-1 and fibrillin-2 will be detectable in the plasma of patients with aortic dissections and aneurysms. We sought to determine whether the plasma fibrillin fragment levels (PFFLs) differ between patients with thoracic aortic pathology and those presenting with nonaortic chest pain. Methods:PFFLs were measured in patients with thoracic aortic aneurysm (n = 27) or dissection (n = 28). For comparison, patients without aortic pathology who had presented to the emergency department with acute chest pain (n = 281) were categorized into three groups according to the cause of the chest pain: ischemic cardiac chest pain; nonischemic cardiac chest pain; and noncardiac chest pain. The PFFLs were measured using a sandwich enzyme-linked immunosorbent assay. Results:Fibrillin-1 fragments were detectable in all patients and were lowest in the ischemic cardiac chest pain group. Age, sex, and the presence of hypertension were associated with differences in fibrillin-1 fragment levels. Fibrillin-2 fragments were detected more often in the thoracic aneurysm and dissection groups than in the emergency department chest pain group (P < .0001). Patients with aortic dissection demonstrated a trend toward increased detectability (P = .051) and concentrations (P = .06) of fibrillin-2 fragments compared with patients with aortic aneurysms. Analysis of specific antibody pairs identified fibrillin-1 B15-HRP26 and fibrillin-2 B205-HRP143 as the most informative in distinguishing between the emergency department and aortic pathology groups. Conclusions:Patients with thoracic aortic dissections demonstrated elevated plasma fibrillin-2 fragment levels (B205-HRP143) compared with patients presenting with ischemic or nonischemic cardiac chest pain and increased fibrillin-1 levels (B15-HRP26) compared with patients with ischemic cardiac chest pain. Investigation of fibrillin-1 and fibrillin-2 fragment generation might lead to diagnostic, therapeutic, and prognostic advances for patients with thoracic aortic dissection.

Objective Tobacco smoke exposure is a major risk factor for aortic aneurysm development. However, the initial aortic response to tobacco smoke, preceding aneurysm formation, is not well understood. We sought to create a model to determine the effect of solubilized tobacco smoke (STS) on the thoracic and abdominal aorta of mice as well as on cultured human aortic smooth muscle cells (HASMCs). Methods Tobacco smoke was solubilized and delivered to mice via implanted osmotic minipumps. Twenty male C57BL/6 mice received STS or vehicle infusion. The descending thoracic, suprarenal abdominal, and infrarenal abdominal segments of the aorta were assessed for elastic lamellar damage, smooth muscle cell phenotype, and infiltration of inflammatory cells. Cultured HASMCs grown in media containing STS were compared to cells grown in standard media in order to verify our in vivo findings. Results Tobacco smoke solution caused significantly more breaks in the elastic lamellae of the thoracic and abdominal aorta compared to control solution (P< .0001) without inciting an inflammatory infiltrate. Elastin breaks occurred more frequently in the abdominal aorta than the thoracic aorta (P < .01). Exposure to STS-induced aortic microdissections and downregulation of α-smooth muscle actin (α-SMA) by vascular smooth muscle cells (VSMCs). Treatment of cultured HASMCs with STS confirmed the decrease in α-SMA expression. Conclusion Delivery of STS via osmotic minipumps appears to be a promising model for investigating the early aortic response to tobacco smoke exposure. The initial effect of tobacco smoke exposure on the aorta is elastic lamellar damage and downregulation of (α-SMA) expression by VSMCs. Elastic lamellar damage occurs more frequently in the abdominal aorta than the thoracic aorta and does not seem to be mediated by the presence of macrophages or other inflammatory cells.

In humans, mutations in the gene for fibrillin‐1 (FBN1) cause both the Marfan syndrome as well as several types of acromelic dysplasias. Musculoskeletal features of the Marfan syndrome (long bone overgrowth and arachnodactyly, joint hypermobility, and a muscle wasting phenotype) are the opposite of those in acromelic dysplasias (short stature and brachydactyly, joint stiffness, and a hypermuscular build). How mutations in the same gene can result in opposite phenotypes is poorly understood. Mutations in FBN2 cause congenital contractural arachnodactyly, which features a mixture of related musculoskeletal features (muscle hypoplasia, contractures of the large and small joints and arachnodactyly).The fibrillins are multifunctional glycoproteins that polymerize into extracellular matrix microfibrils. Although we have associated fibrillin microfibril structure with the opposite musculoskeletal features of Marfan syndrome and acromelic dysplasias, we can only speculate that perturbations in growth factor signaling are involved. To begin to address this gap in our understanding, we hypothesize that direct interactions between fibrillin and cells coordinate growth factor signaling. Therefore, we searched for interactions between fibrillin and cellular receptors.Fibrillins, like Notch receptors and Notch ligands, are composed of multiple tandemly repeated cbEGF‐like domains. Each fibrillin contains 43 cbEGF‐like domains. What if Notch receptors or ligands also bind to fibrillins in the extracellular matrix? Our biochemical data imply that fibrillin interactions with Notch receptors or ligands are just as likely to occur in vivo as interactions between Notch receptors and known Notch transmembrane ligands. Human genetic evidence support significant roles for Notch signaling components in vascular and musculoskeletal diseases, tissues primarily affected in the fibrillinopathies.The current concept of Notch signaling is based strictly on cell‐cell communication. Our investigations have the potential to transform current understanding of context‐dependent Notch signaling by introducing the possibility that Notch signaling components also interact with fibrillin extracellular matrix.Support or Funding Informationgrants from the Shriners Hospitals for Children to Lynn Y. Sakai

Fibrillin-1 is a structural component muscular arteries, and plasma fibrillin-1 fragment levels (PFFLSs) are detectable in patients with aortic dissections and thoracic aortic aneurysms. However, little is known regarding PFFLSs in patients without aortic pathology or in patients with other cardiovascular conditions, thereby limiting its use as a biomarker for aortic pathology. We sought to determine the detectability and concentration of PFFLSs in patients without aortic pathology who present with acute chest pain of cardiac and noncardiac origin. Three hundred patients older than 18 years presenting to the emergency department with chief complaint of chest pain were prospectively enrolled. Patients with aortic pathology, malignancy, or postoperative pain were excluded from analysis. Demographic data, medical comorbidities, and the determined cause of chest pain was determined from the electronic medical record. PFFLSs were determined using a modified sandwich enzyme-linked immunosorbent assay with biotinylated capture antibodies and alkaline phosphatase-conjugated detector antibodies. Patients were categorized into three groups based on the cause of chest pain: group 1, ischemic cardiac chest pain (ICCP), group 2, non-ICCP (NICCP), and group 3, noncardiac chest pain (NCCP). The ICCP group consisted of patients presenting with myocardial infarction or angina. The NCCP included patients presenting with arrhythmia, heart failure, hypertensive urgency, and pericarditis/myocarditis/endocarditis. Differences in categorical variable were assessed using Fisher’s exact test and differences in continuous variables were assessed using Kruskal-Wallis test and Wilcoxon rank-sum test as appropriate. Two hundred sixty-five patients (135 men and 130 women) were included in the analysis. The cause of chest pain was determined to be ICCP in 30 patients, NICCP in 39 patients, and NCP in 196 patients. Detectable PFFLSs were found in 192 (72%) patients and detectability did not differ between genders or diagnosis groups (P = .74). Mean PFFLSs are lower in the ICCP group compared to the NICCP and NCCP groups (0.19, 0.38, and 0.56 μg/mL, respectively; P < .013). PPFLs were lower in ICCP patients with diabetes and there was a trend toward lower PPFLs in men in the ICCP group (Tables I and II). PFFLS are detectible in the majority of all patients presenting with chest pain. PFFLS are lower in patients presenting with ICCP. In patients presenting with chest pain of cardiac origin (ICCP and NICCP), PFFLSs are lower in men and patients with diabetes. Further characterization of PFFLs in patients with and without cardiovascular disease and risk factors has the potential to lead to biomarker development and mechanistic insights into cardiovascular disease development.Table IMean and interquartile values of plasma fibrillin-1 fragment levels by diagnosis groupPFFL (μg/mL)ICCP (n = 30)NICCP (n = 39)NCCP (n = 196)25th percentile00050th percentile0.020.060.0875th percentile0.060.160.23Mean0.19a0.380.56Standard deviation0.830.961.9ICCP, Ischemic cardiac chest pain; NCCP, noncardiac chest pain; NICCP, nonischemic cardiac chest pain; PFFL, plasma fibrillin-1 fragment level.aP < .013. Open table in a new tab Table IIMedian and interquartile plasma fibrillin-1 fragment levels categorized by diagnosis group, gender and presence of diabetes mellitus (DM)PPFL (μg/mL)ICCP (n = 30)NICCP (n = 39)NCCP (n = 196)DM 25th percentile0.0100.03 50th percentile0.030.040.13 75th percentile0.070.380.36 Mean0.04a0.160.55 SD0.040.221.2No DM 25th percentile000 50th percentile0.010.040.07 75th percentile0.040.380.21 Mean0.290.160.57 SD1.10.222.0Male 25th percentile000 50th percentile0.020.060.08 75th percentile0.080.160.22 Mean0.05b0.240.47 SD0.070.381.7Female 25th percentile000 50th percentile0.010.030.07 75th percentile0.040.080.23 Mean0.520.570.4 SD1.51.32.0ICCP, Ischemic cardiac chest pain; NCCP, noncardiac chest pain; NICCP, nonischemic cardiac chest pain; PFFL, plasma fibrillin-1 fragment level; SD, standard deviation.aP < .05 compared to patients with DM in the NICCP and NCCP groups.bP < .07 compared to patients with men in the NICCP and NCCP groups. Open table in a new tab

Tobacco smoke exposure is a major risk factor for aortic aneurysm development. However, the initial aortic response to tobacco smoke, preceding aneurysm formation, is not well understood. We developed a novel model to examine early effect of solubilized tobacco smoke (STS) on the thoracic and abdominal aorta of mice as well as on cultured human aortic smooth muscle cells (HASMC). Nineteen Male C57BL6 mice received an infusion of STS or vehicle via osmotic minipumps for 2 weeks. Six mice were treated with vehicle-containing pumps, eight mice received STS pumps in the peritoneal position and five mice received STS pumps in the subcutaneous position. The thoracic and abdominal aortas were assessed for elastic lamellar damage, smooth muscle cell response, and infiltration of inflammatory cells. Elastin breaks were counted by two independent investigators and differences between groups were assessed using a two-tailed Student t-test. Cultured HASMC were exposed to STS after growth in differentiation media and assessed for α-smooth muscle actin (αSMA) and Mac-2 expression using immunofluorescence and western blot analysis. STS exposure caused increased numbers of breaks in the elastic lamellae of the thoracic and abdominal aorta (Fig 1; P < .001). Elastin breaks occurred more frequently in the abdominal aorta than the thoracic aorta (3.7 breaks per cross-section vs 2.3 breaks per cross-section; P < .001). In vivo, STS exposure caused microdissections of the aorta and a phenotypic switch in vascular smooth muscle cells characterized by downregulation of αSMA (Fig 2) and expression of Mac-2. Cultured HASMC exposed to solubilized tobacco smoke demonstrated loss of αSMA compared to HASMC not exposed to STS. Western blot analysis confirmed the loss of αSMA in HASMC exposed to tobacco smoke. HASMC exposed to STS demonstrated altered, rounded morphology within 2 days of tobacco smoke exposure, correlating with the αSMA loss. In initial experiments, Mac-2 expression was not different between HASMC exposed to STS and those exposed to vehicle. We demonstrate a new model for investigating the initial structural and cellular aortic changes in response to tobacco smoke exposure. Our findings suggest that STS exposure causes aortic elastic lamellar damage and phenotypic changes in VSMC, involving αSMA downregulation and possible Mac-2 upregulation. The VSMC phenotypic changes indicate a loss of normal contractile phenotype and expression of inflammatory markers. Utilizing this model, the mechanism of early tobacco smoke-induced aortic injury and strategies for early detection and prevention of aortic injury prior to clinical disease development can be investigated.Fig 2Immunofluorescence staining of aortic microdissection showing Mac-2 expression (bright green) and α-smooth muscle actin (αSMA) expression (red). The area of microdissection (*) shows increased Mac-2 expression (thick arrow, right) and decreased αSMA expression (thick arrow, left) compared to normal area or aorta showing no Mac-2 expression and normal αSMA expression (thin arrows).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Immunolocalization studies have shown that fibrillin-1 is distributed ubiquitously in the connective tissue space from early embryonic times through old age. When mutated, the gene for fibrillin-1 (FBN1) causes the Marfan syndrome, a common inherited disorder of connective tissue. The multiple manifestations of the Marfan syndrome reflect the known distribution of fibrillin-1 in cardiovascular, musculoskeletal, ocular, and dermal tissues. In this study, a mouse model of Marfan syndrome in which fibrillin-1 is truncated and tagged with green fluorescence was used to estimate the relative abundance of fibrillin-1 in developing tissues. In embryonic tissues, the aorta was the only tissue in which fibrillin-1 green fluorescence was detectable. Other arteries gained detectable fibrillin-1 green fluorescence just after birth. Fibrillin-1 fluorescence was observed at later postnatal times in the lung, skin, perichondrium, tendon, and ocular tissues, while other tissues remained negative. These results indicated that tissues most affected in the Marfan syndrome are the tissues in which fibrillin-1 is most abundant. Focus was placed on the aorta, since aortic disease is life threatening in the Marfan syndrome and fibrillin-1 green fluorescence was most abundant in this tissue. Fibrillin-1 green fluorescence and immunostaining showed that fibrillin-1 is within aortic medial elastic lamellae. Endothelial-specific compared to smooth muscle-specific fibrillin-1 green fluorescence, together with light microscopic analyses of fragmentation of aortic elastic lamellae, demonstrated that smooth muscle cell mutated fibrillin-1 contributed most to progressive aortic fragmentation. However, these studies also indicated that other cells, possibly endothelial cells, also contribute to this aortic pathology. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

The Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury (AKI) guidelines assign the same stage of AKI to patients whether they fulfil urine output criteria, serum creatinine criteria or both criteria for that stage. This study explores the validity of the KDIGO guidelines as a tool to stratify the risk of adverse outcomes in cardiac surgery patients.

[This corrects the article DOI: 10.1371/journal.pone.0181166.].

Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

Rationale: Mutations in fibrillin-1 are associated with thoracic aortic aneurysm (TAA) in Marfan syndrome. Genome-wide association studies also implicate fibrillin-1 in sporadic TAA. Fragmentation of the aortic elastic lamellae is characteristic of TAA. Objective: Immunoassays were generated to test whether circulating fragments of fibrillin-1, or other microfibril fragments, are associated with TAA and dissection. Methods and Results: Plasma samples were obtained from 1265 patients with aortic aneurysm or dissection and from 125 control subjects. Concentrations of fibrillin-1, fibrillin-2, and fibulin-4 were measured with novel immunoassays. One hundred and seventy-four patients (13%) had aneurysms with only abdominal aortic involvement (abdominal aortic aneurysm), and 1091 (86%) had TAA. Of those with TAA, 300 patients (27%) had chronic dissection and 109 (10%) had acute or subacute dissection. Associations of fragment concentrations with TAA (versus abdominal aortic aneurysm) or with dissection (versus no dissection) were estimated with odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, sex, and smoking. Compared with controls, significantly higher percentages of aneurysm patients had detectable levels of fibrillin fragments. TAA was significantly more common (than abdominal aortic aneurysm) in the highest compared with lowest quartile of fibrillin-1 concentration (OR=2.9; 95% CI, 1.6–5.0). Relative to TAA without dissection, acute or subacute dissection (OR=2.9; 95% CI, 1.6–5.3), but not chronic dissection, was more frequent in the highest compared with lowest quartile of fibrillin-1 concentration. Neither TAA nor dissection was associated with fibrillin-2 or fibulin-4. Conclusions: Circulating fibrillin-1 fragments represent a new potential biomarker for TAA and acute aortic dissection.

Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFβ signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.

In humans, mutations in fibrillin-1 result in a variety of genetic disorders with distinct clinical phenotypes. While most of the known mutations in fibrillin-1 cause Marfan syndrome, a number of other mutations lead to clinical features unrelated to Marfan syndrome. Pathogenesis of Marfan syndrome is currently thought to be driven by mechanisms due to haploinsufficiency of wild-type fibrillin-1. However, haploinsufficiency-driven mechanisms cannot explain the distinct phenotypes found in other fibrillinopathies. To test the hypothesis that mutations in fibrillin-1 cause disorders through primary effects on microfibril structure, two different mutations were generated in Fbn1 in mice. One mutation leads to a truncated fibrillin-1 molecule that is tagged with green fluorescent protein, allowing visualization of mutant fibrillin-1 incorporated into microfibrils. In heterozygosity, these mutant mice demonstrate progressive fragmentation of the aortic elastic lamellae and also display fragmentation of microfibrils in other tissues. Fibrillin-2 epitopes are also progressively revealed in these mice, suggesting that fibrillin-2 immunoreactivity can serve as a marker for microfibril degradation. In contrast, a second mutation (in-frame deletion of the first hybrid domain) in fibrillin-1 results in stable microfibrils, demonstrating that fibrillin-1 molecules are not required to be in perfect register for microfibril structure and function and that the first hybrid domain is dispensable for microfibril assembly. Taken together, these results suggest that perturbation of microfibril structure may underlie one of the major features of the Marfan syndrome: fragmentation of aortic elastic lamellae.